Effect of the Heteroaromatic Antenna on the Binding of Chiral Eu(III) Complexes to Bovine Serum Albumin

Complexes to Bovine Serum Albumin

Article

Eﬀect of the Heteroaromatic Antenna on the Binding of Chiral Eu(III)

Chiara De Rosa, Andrea Melchior,* Martina Sanadar, Marilena Tolazzi, Alejandro Giorgetti,

Rui P. Ribeiro, Chiara Nardon, and Fabio Piccinelli*

Cite This: https://dx.doi.org/10.1021/acs.inorgchem.0c01663

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sı Supporting Information

ABSTRACT: The cationic enantiopure (R,R) and luminescent Eu(III) complex

[

Eu(bisoQcd)(H O) ] OTf (with bisoQcd = N,N′-bis(2-isoquinolinmethyl)-trans-1,2-

diaminocyclohexane N,N′-diacetate and OTf = triﬂate) was synthesized and

characterized. At physiological pH, the 1:1 [Eu(bisoQcd)(H O) ] species, possessing

two water molecules in the inner coordination sphere, is largely dominant. The

interaction with bovine serum albumin (BSA) was studied by means of several

experimental techniques, such as luminescence spectroscopy, isothermal titration

calorimetry (ITC), molecular docking (MD), and molecular dynamics simulations

(

MDS). In this direction, a ligand competition study was also performed by using three

clinically established drugs (i.e., ibuprofen, warfarin, and digitoxin). The nature of this

interaction is strongly aﬀected by the type of the involved heteroaromatic antenna in the

Eu(III) complexes. In fact, the presence of isoquinoline rings drives the corresponding

complex toward the protein superﬁcial area containing the tryptophan residue 134

(

Trp134). As the main consequence, the metal center undergoes the loss of one water

molecule upon interaction with the side chain of a glutamic acid residue. On the other hand, the similar complex containing pyridine

rings ([Eu(bpcd)(H O) ]Cl with bpcd = N,N′-bis(2-pyridylmethyl)-trans-1,2-diaminocyclohexane N,N′-diacetate) interacts more

weakly with the protein in a diﬀerent superﬁcial cavity, without losing the coordinated water molecules.

INTRODUCTION

which represents 52% of the protein composition in the

circulatory system, has been broadly studied in light of its very

important physiological and pharmacological functions. For

■

The optical sensing of biologically relevant species is often

performed by lanthanide (Ln(III)) derivatives, mainly Eu(III)

and Tb(III) complexes

−6

example, SA has a limited number of binding sites with high

due to their peculiar properties,

speciﬁcity, which play a crucial role in the transportation and

such as long Ln(III) luminescence lifetime and the possibility

to exploit the so-called antenna effect. Thanks to the ﬁrst

peculiarity, one can use time-gated detection to mitigate the

interference of background ﬂuorescence originating from the

biological sample and hence to isolate the Ln(III)

luminescence. This advantageous feature has been exploited,

for example, to obtain a high signal-to-noise (S/N) ratio by

means of Eu(III)-based probes in the detection of intracellular

delivery of a variety of endogenous and exogenous species.

Human serum albumin (HSA) and bovine serum albumin

(

BSA) are the most extensively studied serum proteins.

Changes of the albumin levels in blood and urine could be

related to several disorders, including liver disease, neoplasia,

nephrotic and diabetic syndrome, and severe dehydration.

Serum albumin can bind lanthanide complexes,

8−20

often

leading to signiﬁcant changes of the Eu(III) and Tb(III)

pH as well as other important bioanalytes, such as

1−26

12,13

luminescence features,

ﬂuorescence.

as well as of the protein

bicarbonate, citrate, lactate, ATP,

and vitamin C.

7−31

Such a piece of evidence oﬀers the

Based on the antenna effect, the luminescence intensity of the

metal ion can be considerably enhanced if the ligand is capable

of strongly absorbing and eﬃciently transferring the UV

excitation to the Ln(III) center. At the same time, high values

of the emission quantum yield are required. High emission

intensity is mandatory when emissive lanthanide complexes are

possibility to detect these proteins by means of optical

spectroscopy techniques. Furthermore, since strong changes in

the ﬂuorescence spectra of the proteins are due to interactions

Received: June 5, 2020

used as tags in bioassays or as optical probes. In addition to

optimal emission properties, a luminescent Ln(III) complex

must interact selectively with a target bioanalyte in a complex

matrix containing competing species, such as proteins. Among

the main proteins in the biological ﬂuids, serum albumin (SA),

XXXX American Chemical Society

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

Article

close to the Trp residues, the regiochemistry can often be

evaluated. In this regard, there are marked similarities between

BSA and HSA in their composition and structure. In fact,

although HSA contains two extra amino acid residues (total

For sensing purposes, these two molecules can be easily

displaced by the target analyte. Since the chirality plays an

important role in the interaction with biomolecules, the

conﬁguration of the two stereogenic carbon atoms of the

ligands was ﬁxed (R,R stereochemistry) so to study

enantiopure complexes.

85) compared to BSA, the carried-out protein sequence

alignment (1AO6.pdb and 3V03.pdb for HSA and BSA,

respectively; Figure S1) pointed out about 76% amino acid

sequence identity. Both proteins consist of two identical

chains, A and B, in turn subdivided into three homologous

evolutionarily related domains (I, II, III) (Figure S2). Each

chain is predominantly in the α-helix form (74%), but HSA has

only one tryptophan residue (Trp-214, buried within the

secondary structure), while BSA contains two, namely, Trp-

The protocol for the synthesis of the new [Eu(bisoQcd)-

(H O) ]CF SO complex is reported in Figure 2.

MATERIALS AND METHODS

■

All commercially available reagents were used as received from

suppliers. Solvents (Sigma-Aldrich) were dried when required using

an appropriate drying agent. Reactions requiring anhydrous

conditions were carried out using Schlenk-line techniques under an

13 (conﬁned within a hydrophobic pocket of the protein in

atmosphere of dry argon. Water refers to high purity H O obtained

the domain II) and Trp-134 (located at the outer surface of the

protein in the domain I). Thus, BSA can be used as a model

from the “Millipore Elix 10” puriﬁcation system. Eu(CF SO )

3 3

(Aldrich, 98%) was stored under vacuum for several days at 80 °C

for HSA, with the advantage of oﬀering two structural probes

and then transferred to the glovebox. All other chemicals were

purchased from Alfa Aesar. Thin-layer chromatography was carried

out on neutral alumina plates (Fluka Analytical) or silica plates

(Sigma-Aldrich) and visualized under UV lamp (254 nm). The

cationic exchange chromatography was performed on SCX cartridges

(1 g) purchased from “Agilent Technologies-Sample Prep solutions”.

contributing with six donor atoms, and two water molecules.

Figure S3 ).

Although the emission of BSA originates mainly from the

two tryptophan residues, tyrosine and phenylalanine side

chains give a partial contribution to the overall BSA

ﬂuorescence. Therefore, signiﬁcant changes of the ﬂuo-

rescence intensity of the BSA protein could be ascribed to

interactions close to the aforementioned domains (I and II),

whereas small changes can be due to interactions occurring in

other protein sites, also near tyrosine and phenylalanine

residues. In addition, the ﬂuorescence intensity is also sensitive

to small modiﬁcations of the local protein secondary

4-Morpholinepropanesulfonic acid (MOPS) buﬀer (purity 99%) was

dissolved in 0.9% w/v NaCl water solution. The pH value was

corrected to the physiological value (7.4) by dropwise addition of

freshly prepared NaOH 10 M. Stock solution of BSA (purity ≥99%,

purchased from Sigma-Aldrich) was freshly prepared and used (2 ×

−4

−1

mol L ) by dissolving the protein in MOPS buﬀer. The solution

was kept in the dark at 4 °C before use.

structure.

Synthesis. The [Eu(bpcd)(H O) ]Cl complex (Figure 1) was

In this work, after the synthesis and physicochemical

characterization of two cationic water-soluble Eu(III)

luminescent complexes, the interaction with BSA was studied

by means of several complementary experimental techniques

synthesized as reported in the literature. Its analogue [Eu(bisoQcd)-

(H O) ](CF SO ) (Figure 1), bearing isoquinoline heteroaromatic

pendants, instead of pyridine ones, was obtained following the

synthetic protocol reported in Figure 2.

Isoquinoline-3-carbaldehyde (2). Under inert atmosphere,

DIBAL-H 1 M in toluene (54.5 mL, 55 mmol) was added dropwise

to a stirred solution of the commercially available methyl ester 1 (6 g,

(

emission spectroscopy, isothermal titration calorimetry) and

biomolecular simulations to obtain information on the aﬃnity,

speciﬁcity, and structural details of the complex/protein

adduct. The investigated complexes (Figure 1) diﬀer in the

nature of the heteroaromatic antenna (pyridine vs isoquino-

line), thus being labeled as [Eu(bpcd)(H O) ]Cl and

32 mmol) in anhydrous toluene (200 mL) at −78 °C. The mixture

was stirred at this temperature for 50 min and then allowed to reach 0

°C. Under argon ﬂow, 1 M HCl (48 mL) was dropwise added and the

resulting suspension was ﬁltered through a Celite pad. The ﬁltrate was

diluted with water (350 mL) and extracted with ethyl acetate (3 ×

[

Eu(bisoQcd)(H O) ]OTf, respectively. In both cases, the

2 2

50 mL). The combined organic phases were washed with saturated

Eu(III) ion is 8-fold coordinated by one ligand molecule,

aqueous NaCl solution, dried over Na SO , and the solvents were

evaporated in vacuo to give 3.44 g of a reddish solid, which was used

in the next step without further puriﬁcation. Yield: 68%, purity: 80%

(

determined by H NMR technique). H NMR (CDCl ) δ (ppm)

.78 [2H, t, J = 7.74 Hz]; 7.99 [1H, d, J = 7.74 Hz]; 8.05 [1H, d, J =

.74 Hz]; 8.36 [1H, s]; 9.35 [1H, s]; 10.25 [1H, -CHO]. C NMR

N,N′-Bis-isoquinolin-3-ylmethyl-cyclohexane-1,2-diamine

(

1R, 2R) (4). Purchased trans-1(R),2(R)-diaminecyclohexane (3)

(

1.55 g, 13.56 mmol) was added at RT to a stirred solution of

isoquinoline-3-carbaldehyde (2.34 g, 14.9 mmol) in anhydrous EtOH

145 mL). The yellowish reaction mixture was stirred to room

(

temperature for 12 h; then, upon cooling at ∼0 °C sodium

borohydride (0.923 g, 24.4 mmol) was directly added (one pot) to

the mixture to get a clear reddish solution. After 5 h, the reaction

mixture was quenched with water and the product was extracted with

dichloromethane. The collected organics were washed with brine

solution and dried over sodium sulfate. Upon removal of the solvent

under reduced pressure, 3.47 g of a yellowish oil was obtained (yield:

Figure 1. Molecular structure of the Eu(III) complexes investigated

here, where bpcd and bisoQcd stand for N,N′-bis(2-pyridylmethyl)-

trans-1,2-diaminocyclohexane N,N′-diacetate and N,N′-bis(2-isoqui-

nolinmethyl)-trans-1,2-diaminocyclohexane N,N′-diacetate, respec-

65%, purity: 80%, determined by H NMR technique).

−

tively. For the sake of clarity, the type of counterionCl in the

H NMR (CDCl ) δ (ppm) 9.17 (s, 2H, isoquinoline), 7.88 (m,

−

case of [Eu(bpcd)(H O) ] and CF SO in the case of [Eu-

2H, isoquinoline), 7.74 (d, J = 8.27 Hz, 2H, isoquinoline), 7.64 (s,

2H, isoquinoline), 7.61 (m, 2H, isoquinoline), 7.51 (m, 2H,

(

bisoQcd)(H O) ] is omitted.

Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Figure 2. Synthetic procedure for the new [Eu(bisoQcd)(H O) ]CF SO compound. (a) Diisobutylaluminum hydride (DIBAL-H) 1 M in toluene

.7 equiv, −78 °C; (b) Isoquinoline-3-carbaldehyde ∼1.1 equiv, absolute ethanol, room temperature, 12 h; (c) NaBH 1.8 equiv, MeOH, room

temperature, 5 h; (d) tert-butyl bromoacetate 2.5 equiv, K CO 2.7 equiv, MeCN, room temperature, 12 h; (e) HCl 6 M aq. 80 °C, 12 h; (f)

Eu(CF SO ) 1 equiv, 2-propanol, room temperature, 12 h.

3 3

isoquinoline), 4.16 (d, J_G_E_M= 13.70 Hz, 2H, methylene), 4.06 (d,

J_G_E_M= 13.70 Hz, 2H, methylene), 2.51 (d, 2H, “CH” cyclohexane),

80 °C. The volume of the reaction mixture was halved under reduced

pressure and the product was puriﬁed by cationic exchange

.22 (m, 2H, cyclohexane), 1.77 (m, 3H, cyclohexane), 1.14−1.41

chromatography SCX (eluent: NH 3 M in MeOH) yielding 1.55 g

(

m, 2H, cyclohexane), 1.00 (m, 1H, cyclohexane). C NMR

CDCl ) δ (ppm) 25.21 (2 CH ), 25.39 (CH ), 31.38 (CH ),

2.42 (CH), 55.35 (CH), 57.60 (2 CH ), 118.02 (−2CH Ar), 126.42

of the desired product as a brownish solid. Yield: 55%. H NMR

(DMSO) δ ppm: 9.31−9.21 (m, 2H), 8.15−8.05 (m, 2H), 8.01−7.89

(m, 2H), 7.80−7.71 (m, 2H), 7.69−7.58 (m, 2H), 4.96−4.71 (m, 1H,

(

−2CH), 126.78 (−2CH), 127.54 (−C), 127.62 (−C−), 130.45

−2CH), 136.40 (−2C−), 152.22 (−2CH), 153.59 (−2CH), 153.85

−C−N), 154.35 (−C−N).

), 4.30−3.63 (m, 4H, CH

), 3.56−3.21 (m, 3H, CH ), 2.30−1.82

(m, 3H, Cy), 1.76−1.50 (m, 3H, Cy), 1.37−0.96 (m, 4H, Cy). UV−

vis spectrophotometry (methanol): ε(322 nm): 5174 M cm . ESI-

MS (Scan ES+; m/z): 573 (100%) = [NaKHL] ; L = bisoQcd .

The triﬂate of the cationic complex [Eu(bisoQcd)(H O) ]

−

−1

2−

{

[2-(tert-Butoxycarbonylmethyl-isoquinolin-3-ylmethylami-

no)-cyclohexyl]-isoquinolin-3-ylmethylamino}-acetic acid

tert-butyl ester (1R, 2R) (5). Amine 4 (0.844 mmol) was dissolved

in a mixture of anhydrous acetonitrile (13 mL) and anhydrous

potassium carbonate (0.315 g, 2.28 mmol) under inert condition

(complex 7) was synthesized as follows. Ligand 6 (60 mg, 0.110

mmol) was dissolved in hot (55 °C) 2-propanol (4 mL). Upon

cooling, Eu(III) triﬂuoromethanesulfonate 98% (66 mg, 0.110 mmol)

was added portion-wise, and a yellowish suspension was formed. After

neutralization with KOH 2 M aq. (pH ∼7), the reaction mixture was

stirred at room temperature for 12 h. The suspension was centrifuged,

the ﬁltrate was concentrated under reduced pressure, and the resulting

solid (105 mg) was puriﬁed by dissolution in methanol, followed by

precipitation in diethyl ether. Upon centrifugation, 28.6 mg of the

desired complex was collected as a beigeish solid. Yield: 32%. UV−vis

(

argon). Then, a solution of tert-butyl 2-bromoacetate (0.312 mL,

.11 mmol) in anhydrous acetonitrile (5 mL) was added dropwise

over 5 min. After stirring over 12 h at room temperature,

dichloromethane was added, and the reaction mixture was washed

with brine solution. The organic phase was evaporated under reduced

pressure to give 0.588 g of a yellowish oil. The product was puriﬁed

by chromatographic column on activated neutral alumina (Al O )

−

−1

using a mixture of cyclohexane (Cy) and ethyl acetate (AcOEt) for

the elution of the product (Rf = 0.35; Cy:AcOEt 7:3 to 1:9). In order

to completely collect the product, a mixture of AcOEt and methanol

spectrophotometry: ε (323 nm): 4510 M cm (water); ε (323

−

−1

nm): 7251 M cm (methanol). ESI-MS (Scan ES+; m/z) in

methanol: 663 (100%); 661 (90%); ([Eu(bisoQcd)] ). 695 (32%);

OH)] ). Elemental Anal. Calcd for

(MW 847.6): C, 43.93; H, 4.04; N, 6.61;

O, 16.99; S, 3.78; found: C, 43.83; H, 3.97; N, 6.52; O,17.08; S, 3.88.

was exploited (AcOEt:MeOH 9:1). 380 mg of a yellowish solid were

693 (21%); ([Eu(bisoQcd)(CH

S·(H O)

obtained. Yield = 72%. H NMR (CDCl ) δ (ppm) 9.04 (s, 1H), 8.10

30EuF

(

s, 1H), 7.98 (m, 1H), 7.88 (d, J = 7.39 Hz, 2H), 7.74 (m, 3H), 7.56

(

m, 4H), 4.26 (m, 6H), 4.04 (m, 1H), 3.62 (m, 1H), 2.79 (m, 1H),

H NMR Spectroscopy. Nuclear magnetic resonance (NMR)

experiments were performed at 298.15 K using a 600 MHz Bruker

Avance III spectrometer equipped with a triple resonance TCI

.31 (m, 2H), 1.83 (m, 3H), 1.46 (s, 18H), 1.28−1.02 (m, 4H).

[2-(Carboxymethyl-isoquinolin-3-ylmethylamino)-cyclo-

hexyl]-isoquinolin-3-ylmethylamino}-acetic acid (1R, 2R)

H bisoQcd, ligand 6 as an ammonium salt). 5 (crude, ∼5.15

{

cryogenic probe. Spectra were usually recorded in CDCl and, unless

(

otherwise stated, chemical shifts are expressed as ppm and referenced

to the internal standard tetramethylsilane (TMS). One-dimensional

mmol) was dissolved in HCl aq (6 M, 80 mL) and stirred for 12 h at

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

Article

NMR spectra were recorded with 32 scans and a spectral width of

the titration cell (optical path: 1 cm). The cell containing the ligand

2019 Hz. All spectra were manually phased and baseline corrected

(C° = 0.045 mM) and an equimolar quantity of Eu(III) was titrated

using TOPSPIN 3.2 (Bruker, Karlsruhe, Germany). Chemical shift,

multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet; b, broad),

coupling constants and integration area are reported.

with NaOH and the pH corresponding to each spectrum was

measured as described above. Formation constants were calculated by

ﬁtting the absorbance values at various wavelengths by using

ESI-MS. Electrospray ionization mass spectra (ESI-MS) were

recorded with a Finnigan LXQ Linear Ion Trap (Thermo Scientiﬁc,

San Jose, CA, USA) operating in positive ion mode. The data

acquisition was under the control of the Xcalibur software (Thermo

Scientiﬁc). A MeOH solution of sample was properly diluted and

injected into the ion source at a ﬂow rate of 10 μL/min with the aid of

a syringe pump. The typical source conditions were: transfer line

capillary at 275 °C; ion spray voltage at 4.70 kV; sheath, auxiliary, and

HypSpec program.

DFT Calculations. The paramagnetic Eu(III) ion has been

replaced by Y(III) which is a suitable substitute. Geometry

optimizations were carried out at DFT level in PCM water using

1,42

the B3LYP

exchange−correlation functional. The 6-31+G(d)

basis set was used for the ligand atoms, while Y(III) ion was described

by the quasi-relativistic small core Stuttgart-Dresden pseudopotential

and the relative basis set. All the ﬁnal structures were checked to be

minima by vibrational analysis. ESP ﬁtting charges were calculated by

sweep gas (N ) ﬂow rates at 10, 5, and 0 arbitrary units, respectively.

Helium was used as the collision damping gas in the ion trap set at a

pressure of 1 mTorr.

using the CHelpG scheme. All calculations were carried out with

Gaussian16.

Elemental Analysis. Elemental analyses were carried out by using

an EACE 1110 CHNOS analyzer.

Fluorometric Titrations. In the titration of the complexes,

progressive amounts of BSA (up to 180 μM for [Eu(bisoQcd)-

(H O) ]OTf and up to 24 μM for [Eu(bpcd)(H O) ]Cl]) were

added to solutions containing the complexes (80 μM). Physiological

solutions were prepared MOPS buﬀered (pH = 7.4) and as isotonic

UV−vis Spectrophotometry. Room temperature electronic

spectra were acquired by a Cary 60 UV−vis spectrophotometer,

equipped with a xenon lamp single source (80 Hz), Czerny−Turner

monochromator, and a photomultiplier (dual silicon diode detectors);

scan rate: 300 nm/min in the 200−800 nm range.

Luminescence and Decay Kinetics. Room temperature

luminescence was recorded by a Fluorolog 3 (Horiba-Jobin Yvon)

spectroﬂuorometer, equipped with a Xe lamp, a double excitation

monochromator, a single emission monochromator (mod. HR320),

and a photomultiplier in photon counting mode for the detection of

the emitted signal. All the spectra were corrected for the spectral

distortions of the setup.

In decay kinetic measurements of Eu(III), a Xenon microsecond

ﬂashlamp was used and the signal was recorded by means of a

multichannel scaling method. True decay times were obtained using

the convolution of the instrumental response function with an

exponential function and the least-squares-sum-based ﬁtting program

systems (0.9% w/v NaCl). After each addition of BSA, UV−vis,

ﬂuorescence, and excitation spectra as well as the Eu(III)- D excited-

state lifetimes were recorded at 298 K.

Titration of BSA was carried out at 298 K. Progressive amounts of

[Eu(bisoQcd)(H O) ]OTf or [Eu(bpcd)(H O) ]Cl up to 200 μM

were added to MOPS-buﬀered physiological solution containing 5

μM of BSA. Integrated areas of BSA ﬂuorescence were analyzed using

the MS-Excel cEST macro. Model robustness was checked by

statistical tests (Akaike information criterion) implemented in the

associated tool Solverstat.

Competitive ﬂuorimetric titration experiments were carried out

involving warfarin (up to 20 μM, in the case of [Eu(bpcd)(H O) ]Cl

and up to 100 μM in the case of [Eu(bisoQcd)(H O) ]OTf) in a

MOPS-buﬀered solution containing BSA-complex adducts (molar

(

SpectraSolve software package).

The decay kinetics of the protein ﬂuorescence was measured at 298

ratio [Eu(bpcd)(H O) ]Cl: BSA = 4:1; [Eu(bpcd)(H O) ]Cl = 80

μM. Molar ratio [Eu(bisoQcd)(H O) ]OTf: BSA = 1:1; [Eu-

2 2

K, using a Chronos BH ISS Photon Counting instrument with

picosecond laser excitation at 280 nm operating at 50 MHz.

Fluorescence decays were then globally ﬁtted with exponential

(bisoQcd)(H O) ]OTf = 80 μM). After each addition of warfarin,

2 2

Eu(III) luminescence emission spectrum was recorded. Similar

experiments were carried out with ibuprofen (up to 0.4 mM) and

digitoxin (up to 1 mM).

functions using Glotaran ver. 1.5.1 software.

Potentiometric and Spectrophotometric Titrations. Stock

solutions of NaOH and HCl were prepared by diluting 1.0 M

standard solutions (Fluka Analytical) in ultrapure water (>18 MΩ·cm,

ELGA Purelab UHQ). The ionic strength of all solutions was adjusted

Isothermal Titration Calorimetry. Titrations were performed

using a TA Instruments TAMIII thermostat equipped with a

nanocalorimeter operating at T = 298.15 K and with a stirring rate

of 50 rpm. The sample cell was ﬁlled with a solution (V = 0.7 mL) of

0.25 mM of BSA in MOPS buﬀer (pH 7.4). Reference cell was ﬁlled

with MOPS buﬀer. The titration syringe contained a solution of the

complex (1.5−3.0 mM) in MOPS buﬀer. In the case of the

[Eu(bisoQcd)(H O) ]Cl complex, it was necessary to add 10% v/v

to 0.1 M with appropriate amounts of NaCl (Riedel-de Haen). Stock

solutions of Eu(III) (80 mM) were prepared by dissolving the

chloride salt (Sigma-Aldrich). The lanthanide content in the stock

solutions was determined by titration with EDTA and xylenol orange

as an indicator in acetate buﬀer. Free acid concentration in

EtOH, due to the low solubility in pure water.

lanthanide solutions were checked by Gran’s method.

The heats of dilution were estimated by using identical injections of

buﬀer solution into the protein. All calorimetric data were corrected

with the heat of dilution by subtracting the blank from the

experiments. At least two independent titration experiments were

performed to conﬁrm consistency. Data analysis was performed using

Protonation constants of the bisoQcd ligand was determined by

acid−base potentiometric titrations. The titration cell was maintained

at constant temperature (T = 298.2 ± 0.1 K) with a circulatory bath

and under an argon radial ﬂux. The electromotive force (emf) data

were collected by using a computer-controlled potentiometer (Amel

Instruments, 338 pH Meter) connected to a combined glass electrode

HypDeltaH.

Molecular Docking. The Y(III) analogues of the two Eu(III)

complexes depicted in Figure 1 were docked against the bovine serum

albumin crystal structure (PDB code: 4F5S) using the Autodock suite

(

Metrohm Unitrode 6.0259.100). The electrode was calibrated before

each experiment by an acid−base titration with standard HCl and

NaOH solutions. The carbonate impurity in solution was checked by

the Gran’s method. Titrations were performed by adding NaOH or

ver. 4.2.6. Two ﬂexible docking experiments for each complex

against two binding sites were performed. These were chosen in order

to include the two tryptophan residues of the structure. The ﬂexible

residues were selected according to a cutoﬀ of 6 Å for each tryptophan

residue: R194, L197, R198, S201, W213, N217, A341, V342, S343,

D450, L454 and E16, E17, F126, K127, A128, D129, E130, K132,

F133, W134, N158, N161, Q165 around W213 and W134,

respectively. Since Autodock suite does not include by default the

Y(III) parameters in its force-ﬁeld, those were manually added to the

parameter’s library. For each Autodock run, a cluster analysis over 100

binding poses were performed.

HCl to ligand solutions (total ligand concentration, C° = 0.72 mM)

by a computer-controlled buret (Metrhom Dosimat 765). At least 100

points were collected for each titration which were afterward

processed with the Hyperquad program to calculate the protonation

constants.

The formation constants for the Eu(III) complex were determined

by spectrophotometric UV−vis/pH titrations. Spectra of the solutions

at variable pH were collected with a Varian Cary 50 spectropho-

tometer equipped with an optical ﬁber probe which was inserted in

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

Molecular Dynamics Simulations. From each docking cluster

analysis, a docked structure from the better cluster was chosen to

perform small molecular dynamics simulations. All MD simulations

were carried out using the GROMACS program, ver. 2016.5. Since

the Y(III) parameters are not included by default in the most

commonly used force-ﬁelds, they had to be included manually into

the force-ﬁeld; Y(III) was treated as an ion. Therefore, the parameters

of each component of the docked structure was prepared diﬀerently.

The topology of the ligand molecule without Y(III) was prepared

using the ANTECHAMER suite, and the charges from the DFT

calculations were included manually. The protein was parametrized

using the pdb2gmx from GROMACS with the AMBER99SB force-

ﬁeld. Y(III) parameters were included manually in the force-ﬁeld.

−

Water molecules and ions (0.154 M of Na /Cl to mimic

physiological conditions) were added to complete the system. The

system was then equilibrated through a complete workﬂow: steepest

descents minimization of 5000 steps, NVT equilibration of 100 ps,

NPT equilibration of 100 ps, and MD production under the NPT

ensemble for 100 ns at room temperature. A pull code with 5000 kJ

mol nm of force between the nitrogen, oxygen, and Y(III) atoms

was included in the MD production to keep the integrity of the

complexes. All calculations were performed within a GPU node

available by the computational platform from the “Centro Piattaforme

Tecnologiche” of the University of Verona.

Figure 3. UV−vis absorption spectra changes during the acid−base

titration (pH 2.4−11.7) of the ligand bisoQcd (0.045 mM) in the

presence of equimolar Eu(III).

−

−2

the three moieties and the steric hindrance may account for the

observed slight diﬀerences. The higher stability of the

[

Eu(bisoQcd)(H O) ] complex with respect to the bQcd

RESULTS AND DISCUSSION

■

derivative is ascribable to the higher basicity of isoquinoline

Ligand Protonation and Complex Formation. The best

ﬁt protonation constants for the bisoQcd ligand (Figure S4)

show the presence of four protonated species, as previously

found for the bpcd counterpart and the quinoline-containing

compared to quinoline. This can be deduced from the

comparison of the energies of the Yttrium counterparts, the

possible isomers [Y(bisoQcd)(H O) ] and [Y(bQcd)-

H O) ] shown in Figure 4 . The isomers with b iso Qcd are

2 2

,35

analogue bQcd (Table 1).

The logK values for the bisoQcd

Table 1. Protonation Constants (logK ) for the bisoQcd

Ligand and Complex Formation Constants (logβ_j)

equilibrium

L+H⇆HL

HL+H⇆H₂L

bisoQcd

bpcd

bQcd CDTA

9.27 ± 0.03

5.86 ± 0.07

3.43 ± 0.07

1.62 ± 0.09

10.53 ± 0.04

9.72

5.87

2.94

2.22

11.19

2.18

9.37

5.85

3.46

1.79

9.97

9.43

6.01

3.68

2.51

H L + H⇆H L

L+Eu⇆EuL

L + Eu ⇆EuL(OH) + H

19.6

with Eu(III) at T = 298.2 K and μ = 0.1 M NaCl. Data for bpcd,

bQcd (N,N′-bis(2-quinolinmethyl)-trans-1,2-diaminocyclohexane

N,N′-diacetate), and CDTA (1,2-diaminocyclohexane-N,N,N′,N′-

tetraacetic acid) are also reported. Charges omitted for clarity.

ligand suggest the presence of two moderately strong acidic

sites and two weakly acidic ones. The ﬁrst protonation

Figure 4. Minimum energy structures of the [Y(L)(H O) ]

constant (logK = 9.22) can be assigned to a tertiary amine, in

complexes (L = bisoQcd, bQcd, bpcd). Hydrogens bonded to the

agreement with those already reported. The spectrophoto-

metric acid−base titration (Figure S5 ) shows that molar

absorbance is almost constant in the 10.5−8.5 pH range and

increases at lower pH with the formation of bi- and

triprotonated species. This indicates that both isoquinoline

C atoms are omitted for clarity.

more stable than the corresponding ones involving the bQcd

ligand by 6.8 and 8.9 kcal mol , respectively. This diﬀerence

seems related with the metal-N bonds which are clearly

−

hetero

moieties are protonated. The last species can be associated

shorter in [Y(bisoQcd)(H O) ] (0.14 and 0.08 Å for the

2 2

with acetate protonation.

trans-O,O and trans-N,N isomers, respectively) and similar to

those found in the (somewhat more stable) complex with bpcd

(Table S1 ). Also, in both cases, the trans-O,O isomer is the

most stable (ΔG(trans-O,O-trans-N,N) = −3.0 and −5.1 kcal

mol for the complex with bisoQcd and bQcd, respectively),

suggesting it as the prevalent form in solution.

The analysis of spectrophotometric data (Figures 3 and S6)

provides a speciation model with only a 1:1 metal−ligand

complex formed ([Eu(bisoQcd)(H O) ] ). Based on the

−

obtained complex formation constants, at physiological pH =

.4 the 1:1 species is largely dominant (>99% calculated for

C_E_u= C_b_i_s_o_Q_c_d= 10 μM, using the constants in Table 1). The

obtained stability is comparable to that found previously for

other complexes (Table 1). The distinct binding strength of

Luminescence. Upon titration of the Eu(III) complexes

with increasing amount of BSA, we observed two opposite

trends. In the case of [Eu(bpcd)(H O) ]Cl, a gradual decrease

Inorg. Chem. XXXX, XXX, XXX−XXX

Figure 5. Evolution of the Eu(III) luminescence emission of (a) [Eu(bpcd)(H O) ]Cl complex (80 μM) upon addition of BSA in the 0−24 μM

concentration range and (b) [Eu(bisoQcd)(H O) ]OTf complex (80 μM) upon addition of BSA in the 0 −180 μ M concentration range, at 298 K.

of the Eu(III) luminescence intensity was detected (Figure

a). Conversely, an enhancement of the lanthanide emission

was observed for the complex [Eu(bisoQcd)(H O) ]OTf

(

Figure 5b).

The spectral ﬁngerprint of the hypersensitive D → F

transition is particularly aﬀected by the addition of BSA. This

behavior suggests a change in the nature of the primary donors

to the Eu(III) center, as the oxygen atom of a water molecule

can be displaced by a functional group of the protein, as

discussed later in the paper.

Based on the overlap between the electronic spectra of BSA

and the [Eu(bpcd)(H O) ]Cl complex in the range 260−290

nm, luminescence spectra of the latter cannot be recorded

upon excitation in the indicated wavelength domain at a BSA

concentration >40 μM (ﬁnal experimental BSA−complex

molar ratio of around 1:3). This is a conditio sine qua non for

respecting the limits of validity of the Lambert−Beer law (Abs

over 3 units). On the contrary, the concentration limit of BSA

was extended to 180 μM (2.25:1 BSA−complex molar ratio)

for the [Eu(bisoQcd)(H O) ]OTf derivative.

Figure 6. Luminescence decay curves of the D excited state of

Eu(III) for the complex [Eu(bisoQcd)(H O) ]OTf complex (80 μM)

upon addition of BSA.

To gain further insights into the interaction, the

the metal center is coordinated by two water molecules (as in

our case) are susceptible to ligand displacement by competitive

binding to endogenous serum anions, such as carbonate, or

luminescence decay of the D excited state was recorded.

Figure 6 shows the collected proﬁles for the complex involving

bisoQcd as a ligand at diﬀerent protein concentrations. Under

the same experimental conditions, the compound

protein carboxylic acid residues. Based on these consid-

erations, the Eu(III) luminescence intensity increases, as well

as the quantum yield. On the contrary, the protein/complex

interaction seems not to aﬀect the number of water molecules

bound to the metal ion in the [Eu(bpcd)(H O) ]Cl complex.

[

Eu(bpcd)(H O) ]Cl showed no signiﬁcant change in the

2 2

observed lifetime (ca. 0.30 ms).

The blue and green decay curves cannot be ﬁtted by a single

exponential function when BSA and the complex are both

present in solution, likely due to the presence of more than one

emitting species (complex/protein adducts) in solution. In

The decrease of the Eu(III) emission intensity could be in part

due to the Inner Filter Eﬀect (IFE). IFE is an alternative

signal transduction mechanism that relies on the reduction of a

ﬂuorophore’s emission, caused by preventing its excitation by

absorbing light at its excitation wavelength by other

chromophores: in the case of [Eu(bpcd)(H O) ]Cl complex,

such cases, the values of the 1/e folding time is given instead of

the observed lifetime. The increase of the D excited state

lifetime upon addition of the protein [0.28 ms for the free

complex, 0.34 and 0.50 ms after the addition of BSA (24 μM

and 180 μM, respectively)] can be attributed to a lower

eﬃciency of the multiphonon relaxation process (i.e., process

able to quench the excited state of the lanthanide ion in a

the excitation wavelength of the complex (around 270 nm) is

partially absorbed by BSA, which presents an absorption

maximun around 280 nm. On the other hand, the

aforementioned decrease of the Eu(III) emission intensity

could be also connected to a less eﬃcient ligand-to-metal

energy transfer. In this regard, generally the interaction with

BSA involves the tryptophan residues in domains I and II

7,58

nonradiative way).

In fact, in the binding site some

functional groups of the protein may replace water molecules

in the inner coordination sphere of the metal ion. In this

regard, literature data conﬁrm that Ln(III) complexes wherein

(Trp-134 and Trp-213; Figure S3 ), which could bind the

Inorg. Chem. XXXX, XXX, XXX−XXX

Figure 7. Evolution of the ﬂuorescence spectrum of BSA (5 μM solution) upon addition of (a) [Eu(bpcd)(H O) ]Cl and (b)

[

Eu(bisoQcd)(H O) ]OTf. On the right, the integrated area of each spectrum (○) vs total complex concentration together with the ﬁt (line)

2 2

obtained with the formation constants in Table 2.

metal ion instead of the antenna. Despite this observation, the

excitation spectrum of [Eu(bpcd)(H O) ]Cl shows the

with previous considerations, the R value remains basically

constant (around 1.55) during BSA additions for the

[Eu(bpcd)(H O) ]Cl complex. Moreover, the recorded

presence of a peak around 265 nm which undergoes a blue-

shift toward 262 nm upon addition of the protein (Figure S7a).

This peak is indicative of the luminescence sensitization of

Eu(III) by pyridine rings; thus, we can rule out the

involvement of the tryptophan rings in the sensitization

mechanism, as their typical excitation peak around 280 nm is

absent. Instead, the aforementioned blue-shift could be due to

a change of the energy of the electronic levels of the pyridine

antenna, thus being detrimental to the eﬃciency of the energy

transfer.

excitation spectra for [Eu(bisoQcd)(H O) ]OTf (Figure

S7b ) reveal no or negligible energy transfer mechanisms

involving chromophoric protein groups (i.e., no excitation peak

around 280 nm in the spectrum).

In order to gain more insights into the protein/complex

interaction mechanism, we also investigated the evolution of

the protein ﬂuorescence upon addition of the Eu(III) complex.

In this case, the limits of validity of the Lambert−Beer law

cover both a wider complex concentration range (up to 200

μM) and a larger complex/protein molar ratio (up to 40:1). In

both cases, we observed a decrease of the ﬂuorescence

intensity, which is particularly clear for the derivative with

bisoQcd as a ligand (Figure 7).

The unusual behavior of the [Eu(bpcd)(H O) ]Cl complex

points out a diﬀerent nature of the protein/complex

interaction, which should primarily involve the organic ligand

rather than the metal ion. Vice versa, the involvement of the

Eu(III) coordination sphere of the [Eu(bisoQcd)(H O) ]

In addition, the maximum emission peak of the protein

complex in the interaction with the protein was also conﬁrmed

(

around 335 nm) undergoes a 10 nm red-shift. Therefore, the

by the change in the asymmetry ratio value

corresponding transition (π* → π) occurs in a locally more

polar medium. In other words, the BSA binding to the

[Eu(bisoQcd)(H O) ]OTf complex leads to a protein

I( D → F )

R =

I( D → F )

conformational change, in turn decreasing the rigidity of the

Trp environment and/or exposing such an amino acid residue

indicative of the degree of asymmetry of the coordination

polyhedron around the Eu(III) ion, which passes from 3.80 at

the beginning of the titration to 4.20 at the end. In agreement

to a more hydrophilic environment. Since upon excitation

around 280 nm we mainly excite Trp side chains and to a

lesser extent the tyrosine residues, the signiﬁcant change of

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

Article

the ﬂuorescence intensity of the protein is indicative of an

interaction taking place close to the Trp residues discussed

above. On the contrary, the small change of ﬂuorescence

7a), where a small change in the intensity of the protein

ﬂuorescence was observed upon titration with the complex.

This result suggests that IFE does not aﬀect dramatically the

protein ﬂuorescence in the presence of [Eu(bpcd)(H O) ]Cl.

intensity detected for [Eu(bpcd)(H O) ] predicts an

interaction located in a diﬀerent protein site.

For the [Eu(bisoQcd)(H O) ] complex, ITC data (Figure

2 2

The ﬂuorescence decrease by our complexes can result from

a variety of phenomena, such as collisional (or dynamic)

8b) support the presence of 1:1 + 1:2 protein−complex

adducts, established by a stronger interaction compared to the

other derivative. When both logK and ΔH were set as free

parameters in ITC data ﬁtting, very large errors were obtained;

hence, the values are not reported in Table 2.

quenching and static quenching. Regarding the former (a

bimolecular process), upon contact of BSA with some

molecule in solution (e.g., the Eu(III) complex) via an

inelastic collision, the ﬂuorophore reaches its excited state,

then deactivated by heat release. Conversely, in the static case

the ﬂuorophore can form nonﬂuorescent adducts with the

quencher. In order to discriminate between static or dynamic

quenching, we measured the lifetime of the protein excited

state. The ﬂuorescence decay of BSA was best ﬁtted with a

biexponential function, and the corresponding averaged

Ligand Competition Studies. To identify the BSA sites

involved in the interaction with the Eu(III) complexes,

ﬂuorimetric titrations using site-selective competitive ligands

(the clinically established drugs ibuprofen, warfarin, and

66−68

digitoxin)

were carried out. Warfarin is selective for the

external site (domain I) where Trp-134 residue is present,

while ibuprofen for the domain II, associated with the inner

Trp-213 residue (Figure S2). Digitoxin shows a selective

interaction with the domain III, not ﬂuorescent, and with

lifetimes, ⟨τ ⟩, remained unaltered upon addition of both

complexes (around 7.5 ns). This indicated that the quenching

of the ﬂuorescence follows a static mechanism and a ground-

state complex between BSA and each Eu(III) complex should

which our complexes seem not to interact.

Upon addition of warfarin to the solution containing the

BSA/[Eu(bpcd)(H O) ]Cl adduct, a decrease of the Eu(III)

be present in solution.

The ﬂuorescence data were analyzed by the well-known

luminescence intensity within the experimental error was

Stern−Volmer plot. As shown in Figure S8, a linear

detected. Since the BSA−warfarin adduct is associated with a

correlation of the ﬂuorescence intensities F /F with the

logK = 5.07, higher than the one calculated for the BSA/

concentration is undoubtedly observed only in the case of

[Eu(bpcd)(H O) ] , there is no competition between warfarin

[

Eu(bpcd)(H O) ]Cl complex. The determined Stern−

and such a complex for the domain I. Likewise, the

[Eu(bpcd)(H O) ]Cl complex does not interact with the

−

Volmer constant K of this compound is 2089 M . The

analysis of BSA emission data, by means of the program MS-

Excel cEST macro, provided best-ﬁtting models (Figure 7,

right) which correspond to the formation of a 1:1 adduct for

domain II, as the Eu(III) luminescence intensity does not

change upon addition of ibuprofen to the BSA/complex

adduct. On the contrary, the addition of warfarin to the BSA

the [Eu(bpcd)(H O) ]Cl complex (Figure 7a) and of 1:1 +

adduct(s) with [Eu(bisoQcd)(H O)] resulted in a signiﬁcant

:2 species for the original complex [Eu(bisoQcd)(H O) ]

2 2

decrease (around 85%) of the Eu(III) luminescence intensity

(Figure 7b). The obtained adduct formation equilibrium

(

Figure 9).

constants (logK) are reported in Table 2. Interestingly, the

This behavior, opposite the one highlighted in Figure 5b,

points out the Eu(III) complex replacement by warfarin at site

I (Trp 134). However, both ﬂuorimetric and calorimetric

titrations suggest that [Eu(bisoQcd)(H O) ]OTf can interact

Table 2. Formation Constants for the Complex/BSA

Adducts Obtained from Fluorimetric and ITC Titrations

with BSA in an additional position. Since the Eu(III)

luminescence intensity does not change for this compound

upon addition of both ibuprofen and digitoxin, the second

interaction site is diﬀerent from those of the two here studied

drugs.

In the case of warfarin and ibuprofen, it was checked that

each drug interacts with the protein by monitoring, upon

titration, the BSA ﬂuorescence stemming from the adduct with

the complexes. The addition of these drugs results in a

signiﬁcant decrease of the protein ﬂuorescence intensity. In

order to better understand the nature of the protein/complex

interactions, molecular docking and molecular dynamics

logK

ΔH

₍_k_J_m_o_l₋1

)

L =

bpcd

reaction

Fluorimetry

logK ITC

BSA + EuL ⇋

BSA[EuL]

3.7 ± 0.6

3.9 ± 0.2

3.6 ± 0.4

3.61 ± 0.07

−13.1 ± 0.1

−16 ± 3

bisoQcd BSA + EuL ⇋

BSA[EuL]

BSA[EuL] +

−32 ± 4

EuL ⇋

BSA[EuL]

LogK_s_vvalue reported above for the [Eu(bpcd)(H O) ]Cl

complex is in good agreement with the formation constants for

the complex/BSA adduct reported in Table 2 (LogK = 3.32 ±

simulations were performed.

.02; LogK = 3.7 ± 0.6). As expected for the static quenching

[Y(bpcd)(H

]

Complex. When the computationally

2 2

regime, the Stern−Volmer quenching constant is identiﬁed

equivalent [Y(bpcd)(H O) ] complex is docked close to the

with the adduct association constant.

sites I and II (associated with the presence of Trp134 and

Trp213, respectively), it does not remain in these binding sites.

It explores all the protein surfaces after the ﬁrst nanoseconds of

Isothermal Titration Calorimetry. As initially diﬀerent

models provided a reasonable ﬁt of the ﬂuorimetric data and

these data could be aﬀected by the inner ﬁlter eﬀect (IFE), the

BSA−complex interaction was also studied by a diﬀerent

experimental technique such as isothermal titration calorimetry

simulation. Then, [Y(bpcd)(H O) ] interacts with a super-

ﬁcial cavity where it spends half the simulation time (50 ns)

(Figures 10 and S9 ).

(

ITC). For the [Eu(bpcd)(H O) ]Cl (Figure 8a) a 1:1 model

The amino acid residues located at the external surface of

this cavity and therefore likely involved in the interaction with

the pyridine-based complex consist of several groups arising

from four residues of phenylalanine (F227, F308, F325, and

well ﬁts the heat data, resulting in logK = 3.61 and a negative

formation enthalpy (ΔH) (Table 2). This value of logK agrees

with the constant obtained from luminescence data (Figure

Inorg. Chem. XXXX, XXX, XXX−XXX

exchanged/total moles of added reactant) vs complex/BSA molar ratio.

Figure 8. Calorimetric titrations of (a) BSA (0.25 mM) with [Eu(bpcd)(H O) ]Cl (1.5 mM). Solvent: aqueous solution of MOPS, 13 mM; pH =

.4. Final Eu/BSA molar ratio = 2.14. (b) BSA (0.25 mM) with [Eu(bisoQcd)(H O) ]OTf (3 mM). Solvent: aqueous solution of MOPS 13 mM

2 2

with EtOH 10% v/v; pH = 7.4. Final Eu/BSA molar ratio: 4.8. On the right, the experimental (○) and calculated (line) Q_c_u_m(cumulative heat

particular, the positive charge of the complex can give rise to

a Coulombic interaction with the negatively charged aspartic

and glutamic acid side chains. The pyridine rings of the ligand

can interact by means of van der Waals contacts with the side

groups of aromatic amino acids listed above. As mentioned in

the Introduction, since such amino acids (phenylalanine and

tyrosine) contribute to the protein ﬂuorescence to a small

extent, their involvement in the interaction with the complex

[

Eu(bpcd)(H O) ] could aﬀect only marginally the ﬂuo-

rescence spectrum of BSA (Figure 7a).

It is worth noting that upon interaction the metal ion does

not lose the bound water molecules (two), as shown in the

Figure S10a , where the graphical representation of the radial

distribution function is reported, as a function of the

O(water)−Y distance. In fact, the number of water molecules

in the inner coordination sphere of the metal ion (at 2.6 Å) is

around two, both in the simulation in bulk water and upon

interaction with the protein. This is in good agreement with

the analysis of the Eu(III) luminescence decay curves

discussed in the previous section.

Figure 9. Luminescence of the complex [Eu(bisoQcd)(H O) ](OTf)

(

80 μM) interacting with BSA (80 μM) upon titration with warfarin

up to 100 μM); room temperature; solvent: aqueous solution of

MOPS, 13 mM; pH = 7.4.

[

Y(bisoQcd)(H O) ] Complex. [Y(bisoQcd)(H O) ]

2 2 2 2

shows a selective interaction with the outer binding site

containing the Trp residue 134, as it remains in this protein

superﬁcial area through all the simulation time (100 ns). On

F329) and two residues of tyrosine (Y318 and Y331). In this

superﬁcial area also, ﬁve negatively charged carboxylic groups

are present [from three aspartic acids (D307, D311, D313)

and two glutamic acids (E320, E332); Figure10b]. This piece

the contrary, no interaction occurs with the buried Trp213, as

of evidence is in line with the possible interactions that

in the case of the [Y(bpcd)(H

] derivative. From the

[

Y(bpcd)(H O) ] can establish with the protein. In

thermodynamic point of view, based on the binding energy

Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Figure 10. (a) BSA superﬁcial interaction site with the [Y(bpcd)(H O) ] complex. The electrostatic potential is represented (electronegative

cavities are red colored) (left) as well as the distance between the interacting complex and the buried Trp 213 (right). (b) Close-up on the

[

Y(bpcd) (H O) ] that lies on the superﬁcial cavity and representation of the amino acid residues surrounding the complex (distances averaged

2 2

along the simulation).

analysis obtained from docking (Figure S11), this inner site is

not suitable for both studied complexes.

If we focus on the superﬁcial binding site, the distance

between Y(III) and Trp134 was constant (around 6.8 Å) over

all simulation time (Figure S12 ). In particular, we noticed the

presence of a “non-conventional” hydrogen bond involving

the aromatic ring of Trp 134 as H-bond acceptor and a C−H

bond of the isoquinoline ring as donor (Figure S13). The

phenomenon of the “CH/π interaction” in organic and

biological chemistry has been explored and reviewed by Nishio

et al. in a very interesting way.

These results are in perfect agreement with the outcomes

associated with the analysis of the BSA ﬂuorescence upon

interaction with the complex and the competitive titration

experiments with the drug warfarin. In addition, one

coordinated water molecule is displaced by one monodentate

carboxylic group of the glutamic acid residue 17 (E17) (Figure

Figure 11. Snapshot of the interaction site of BSA with the

[Y(bisoQcd)(H O)] complex.

1).

In addition, this ﬁnding is in agreement with the increase of

una ﬀected by the interaction with protein (Figures S14 and

S15). Focusing on the complex geometry, only small

the observed luminescence lifetime of Eu(III) during the

titration of [Eu(bisoQcd)(H O) ] complex with the protein

diﬀerences in the orientation of the heteroaromatic fragments

(Figure 6). In fact, the removal of one water molecule from the

are noticed for [Y(bisoQcd)(H O)] upon interaction with

inner coordination sphere of Eu(III) reduces the multiphonon

relaxation process. A radial distribution analysis (Figure S10b)

conﬁrmed that, while in bulk water the metal ion has two water

molecules in its coordination sphere (at a distance of ∼2.5 Å),

when the complex is docked into the binding site close to

Trp134, the ion just retains one water molecule. Interestingly,

an inspection of the Y coordination environment for both

complexes revealed that the bond distances between the metal

ion and the donor atoms of the ligands are in practice

BSA (Figure S16 ). In contrast, a signiﬁcant change in the

orientation of the pyridine rings was detected in the case of the

[Y(bpcd)(H O) ] complex. In this case, the lone pair of one

pyridine nitrogen is not pointing precisely toward the metal

ion (see Figure S17 for details).

To sum up, molecular docking calculations led to results in

agreement with those achieved via spectroscopy. In fact, on

one hand the interaction of the [Y(bisoQcd)(H O) ] complex

with the protein mainly aﬀects the environment of the metal

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

The Supporting Information is available free of charge at

Article

ion (i.e., displacement of a water molecule by E17, acting as a

interaction with the protein and, at the same time, take

advantage of the relaxivity increase due to the adduct

formation with the protein.

monodentate O-donor ligand). On the other hand, the

coordination sphere of [Y(bpcd)(H O) ] remains the same

before and after the interaction with the metal center playing

no key role. The interaction is instead mediated by the ligand

scaﬀold, establishing various interactions with the model

protein based on diﬀerent topological (morphological and

chemical) complementarities.

Finally, this work paves the way to future studies aimed at

designing and developing new molecular luminescent chiral

probes wherein the nature and the scaﬀold size of the

multidentate ligand as well as the stability of the M−L bonds

play a role of paramount importance to enable or reduce the

accessibility to the Ln(III) center.

At this stage, it is useful to recall the spin selection rules

underlying the energy exchange mechanism. The eﬃciency

of this processgoverning a nonradiative energy transfer and

involving the triplet state of a ligand and the excited states of

lanthanide ionsis optimal if (i) the energy overlap of the

absorption band of the donor (D) and the emission band of

the acceptor (A) is high; (ii) the D−A distance is small; and

ASSOCIATED CONTENT

Supporting Information

https://pubs.acs.org/doi/10.1021/acs.inorgchem.0c01663.

■

sı

Listings of structural data of BSA and HSA proteins,

potentiometric and acid−base spectrophotometric data,

luminescence excitation data, Stern−Volmer plots, radial

distribution functions, and molecular docking data

(

iii) the orientation of the orbitals involved in the mechanism

is such that their overlap is good. Since the orbitals of the

pyridine rings should be involved in such a mechanism, the

observed change in their orientation upon protein interaction

could worsen the orbital overlap discussed at the point (iii)

and, in turn, the eﬃciency of the exchange mechanism. This

conclusion is also supported by spectroscopy: apart from the

IFE, the deterioration of the ligand-to-metal energy transfer

eﬃciency could contribute to the decrease of the luminescence

emission intensity of the [Eu(bpcd)(H O) ]Cl complex during

(

coordination distances and geometries) (PDF )

AUTHOR INFORMATION

Corresponding Authors

■

Fabio Piccinelli − Luminescent Materials Laboratory,

Department of Biotechnology, University of Verona and INSTM

- UdR Verona, 37134 Verona, Italy; orcid.org/0000-0003-

the titration with BSA (Figure 5a).

349-1960; Email: fabio.piccinelli@univr.it

CONCLUSIONS

The new [Eu(bisoQcd)(H O) ] complex was synthesized, in-

depth characterized, and its solution behavior investigated. The

Andrea Melchior − Laboratory of Chemical Technologies,

Polytechnic Department of Engineering and Architecture,

University of Udine, 33100 Udine, Italy; orcid.org/0000-

■

002-5265-1396 ; Email: andrea.melchior@uniud.it

stability is slightly higher than that of the previously studied

[

Eu(bQcd)(H O) ] counterpart, due to the larger basicity of

Authors

the isoquinoline compared to quinoline. The speciation

showed that the EuL complex is the only species present at

physiological pH, thus making it a good candidate for

luminescent sensing of bioanalytes.

Chiara De Rosa − Luminescent Materials Laboratory,

Department of Biotechnology, University of Verona and INSTM

UdR Verona, 37134 Verona, Italy

Martina Sanadar − Laboratory of Chemical Technologies,

Polytechnic Department of Engineering and Architecture,

University of Udine, 33100 Udine, Italy

Marilena Tolazzi − Laboratory of Chemical Technologies,

Polytechnic Department of Engineering and Architecture,

University of Udine, 33100 Udine, Italy

Alejandro Giorgetti − Applied Bioinformatics Laboratory,

Department of Biotechnology, University of Verona, 37134

Verona, Italy; orcid.org/0000-0001-8738-6150

Rui P. Ribeiro − Applied Bioinformatics Laboratory,

Department of Biotechnology, University of Verona, 37134

Verona, Italy

The interaction of [Eu(bisoQcd)(H O) ] and [Eu(bpcd)-

(

H O) ] with BSA, by us conceived as a potential competitor,

2 2

was studied by ITC and by analysis of the lanthanide and

protein emission spectra. Experimental data revealed a

markedly diﬀerent behavior of the two complexes. [Eu(bpcd)-

(

H O) ] can form only a 1:1 adduct on an external pocket of

the protein as suggested by MD with no involvement of the

speciﬁc sites of three drugs explored here via competitive

titrations. The luminescence decay remains unchanged when

the complex interacts with the protein, thus indicating a mere

structural role for the metal center (M). The M−L bonds

proved strong enough to keep the pharmacophore-like scaﬀold

in ﬁxed positions, thus pointing to speciﬁc amino acid residues.

Chiara Nardon − Luminescent Materials Laboratory,

Department of Biotechnology, University of Verona and INSTM

On the contrary, [Eu(bisoQcd)(H O) ] directly involves the

UdR Verona, 37134 Verona, Italy

Eu(III) ion during the interaction with the protein by

coordination to a glutamate side chain (E17) in parallel with

the loss of one water molecule in the ﬁrst coordination sphere,

thus increasing the luminescence lifetime. It can establish two

types of adduct, namely, with 1:1 and 1:2 protein/complex

stoichiometry. At least one occurs in the Trp 134 region

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.inorgchem.0c01663

Notes

The authors declare no competing ﬁnancial interest.

(

protein domain I), as highlighted by the warfarin displace-

ment.

It is worth highlighting that the indications obtained could

ACKNOWLEDGMENTS

■

The authors thank the Italian Ministry of education, university

and research for the received funds (PRIN (Progetti di Ricerca

di Rilevante Interesse Nazionale) project “CHIRALAB”, grant

no. 20172M3K5N). The authors gratefully thank also the

Facility “Centro Piattaforme Tecnologiche” of the University

be exploited in the development of Gd(III)-based blood-pool

magnetic resonance imaging (MRI) contrast agents. Indeed,

similarly to the two Eu(III) complexes studied here, Gd(III)

analogues should retain at least one water molecule upon

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

Article

of Verona for access to the Fluorolog 3 (Horiba-Jobin Yvon)

spectroﬂuorometer and to the computational platform.

Funding from the University of Verona is gratefully acknowl-

edged.

Exchange Dynamics, and Human Serum Albumin Binding. Inorg.

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Chem. Commun. 2017, 53, 6724−6727.

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